Medicament for therapeutic treatment and/or improvement of sepsis

ABSTRACT

A medicament for therapeutic treatment and/or improvement of sepsis in a patient with severe sepsis accompanied with one or more organ dysfunctions, wherein a value of International Normalized Ratio (INR) of a plasma specimen obtained from said patient is more than 1.4, which comprises thrombomodulin as an active ingredient.

TECHNICAL FIELD

The present invention relates to a medicament for therapeutic treatmentand/or improvement of sepsis in a severe septic patient.

BACKGROUND ART

Sepsis is a systemic inflammatory response syndrome (SIRS) induced byinfection. Specifically, sepsis is defined as a pathological conditionthat meets, in addition to the presence of infection, two or more of theSIRS items ((1) body temperature >38° C. or <36° C., (2) heartrate >90/minute, (3) respiration rate >20/minute, or PaCO₂<32 torr, and(4) leucocyte count >12,000/μL or <4000/μL, or immatureleucocytes >10%). Although presence of bacteria in blood (bacteremia)has been significantly focused so far, bacteria-positive result of bloodculture is not necessarily required according to the above definition.Among sepsis, a condition presenting organ dysfunction, organhypoperfusion, or hypotension is called severe sepsis. The organhypoperfusion or abnormal perfusion includes lactic acidosis, oliguria,mental clouding, and the like. Among the severe sepsis, a conditioncontinuously presenting hypotension despite of sufficient load of fluidtherapy is called septic shock (Non-patent document 1). It is consideredthat the circulatory failure observed in these pathological conditionsis caused by malfunction of the sympathetic nervous system or a mediatorreleased from neutrophiles and the like, and the organ dysfunction iscaused by tissue hypoxia (dysoxia).

Thrombomodulin has been known as a substance that acts to specificallybind to thrombin so as to inhibit the blood coagulation activity ofthrombin, and at the same time, exerts anticoagulant activity so as tosignificantly promote the ability of thrombin to activate Protein C. Ithas also been known that thrombomodulin exerts to prolong the clottingtime by thrombin, or suppresses platelet aggregation by thrombin.Protein C is a vitamin K-dependent protein that plays an important rolein a blood coagulation and fibrinolysis, and activated by the action ofthrombin to be converted as activated Protein C. It has been known thatthe activated Protein C inactivates activated blood coagulation factor Vand activated blood coagulation factor VIII in vivo, and is involved ingeneration of a plasminogen activator having thrombolytic action(Non-patent document 2). Accordingly, it has been considered thatthrombomodulin promotes the activation of Protein C by thrombin, andtherefore is useful as an anticoagulant or a thrombolytic agent. It hasalso been reported that, in an animal experiment, thrombomodulin iseffective for therapy or prophylaxis of diseases associated withhypercoagulable state (Non-patent document 3).

Thrombomodulin was first discovered and obtained as a glycoproteinexpressed on the vascular endothelial cells of various animal speciesincluding humans, and then successfully cloned. Specifically, a gene ofa human thrombomodulin precursor including a signal peptide was clonedfrom a human lung cDNA library by genetic engineering techniques and theentire gene sequence of thrombomodulin was analyzed, and as a result, anamino acid sequence consisting of 575 residues containing a signalpeptide (in general, 18 amino acid residues are exemplified) wasrevealed (Patent document 1). It is known that a mature thrombomodulin,from which the signal peptide is cleaved, is composed of 5 regions,namely, an N-terminal region (amino acid residues 1 to 226, thesepositions are defined under an assumption that the signal peptideconsists of 18 amino acid residues, and the same shall apply to thefollowing descriptions), a region having six EGF-like structures (aminoacid residues 227 to 462), an O-linked glycosylation region (amino acidresidues 463 to 498), a transmembrane region (amino acid residues 499 to521), and an cytoplasmic region (amino acid residues 522 to 557), fromthe N-terminal side of the mature peptide. It is also known that apartial protein having the same activity as that of the entire lengththrombomodulin (i.e., a minimal active unit) is mainly consisting of the4th, 5th, and 6th EGF-like structures from the N-terminal side in theregion having six EGF-like structures (Non-patent document 4).

The entire length thrombomodulin is hardly dissolved in the absence of asurfactant, and addition of a surfactant is essential for manufacturingan entire thrombomodulin preparation. A soluble thrombomodulin is alsoavailable that can be fully dissolved even in the absence of asurfactant. The soluble thrombomodulin may be prepared by removing atleast a part of the transmembrane region or the entire transmembraneregion. For example, it has been confirmed that a soluble thrombomodulinconsisting of only 3 regions, namely, the N-terminal region, the regionhaving six EGF-like structures, and the O-linked glycosylation region(i.e., a soluble thrombomodulin having an amino acid sequence consistingof amino acid residues 19 to 516 of SEQ ID NO: 9) can be obtained byapplying recombination techniques, and that the resulting recombinantsoluble thrombomodulin has the same activity as that of an entirethrombomodulin (Patent document 1). Some other reports are alsoavailable regarding soluble thrombomodulins (Patent documents 2 to 9). Ahuman urine-derived soluble thrombomodulin and the like are alsoexemplified as native thrombomodulins (Patent documents 10 and 11).

As recognized in many cases, as a result of spontaneous mutations ormutations occurring at the time of obtainment, polymorphic mutationshave been found in the human genes. At present, thrombomodulin proteinsin which the amino acid at the position 473 of human thrombomodulinprecursor having the amino acid sequence consisting of 575 amino acidresidues is converted to Val or Ala have been identified. In thenucleotide sequence encoding the amino acid sequence, this variation ofamino acid residue corresponds to mutation to T or C at the position1418 (Non-patent document 5). However, the two types of thrombomodulinsare completely identical in terms of their activity and physicochemicalproperties, and it can be considered that they are substantiallyidentical.

It has been reported that thrombomodulin is effective for a therapeutictreatment of disseminated intravascular coagulation (henceforth alsoreferred to as DIC) (Non-patent document 6). As for use ofthrombomodulin, in addition to the aforementioned use, thrombomodulin isexpected to be used in therapeutic and prophylactic treatments ofvarious diseases such as acute coronary syndrome (ACS), thrombosis,peripheral vessel obstruction, obstructive arteriosclerosis, vasculitis,functional disorder occurring after heart surgery, complication causedby organ transplantation, angina pectoris, transient ischemic attack,toxemia of pregnancy, diabetes, liver VOD (liver veno-occlusive disease,e.g., fulminant hepatitis, veno occlusive disease of liver occurringafter bone marrow transplantation), and deep venous thrombosis (DVT),and further, sepsis and adult respiratory distress syndrome (ARDS)(Patent document 12).

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: Japanese Patent Unexamined Publication (Kokai)    No. 64-6219-   Patent document 2: Japanese Patent Unexamined Publication No.    5-213998-   Patent document 3: Japanese Patent Unexamined Publication No.    2-255699-   Patent document 4: Japanese Patent Unexamined Publication No.    3-133380-   Patent document 5: Japanese Patent Unexamined Publication No.    3-259084-   Patent document 6: Japanese Patent Unexamined Publication No.    4-210700-   Patent document 7: WO92/00325-   Patent document 8: WO92/03149-   Patent document 9: WO93/15755-   Patent document 10: Japanese Patent Unexamined Publication No.    3-86900-   Patent document 11: Japanese Patent Unexamined Publication No.    3-218399-   Patent document 12: WO03/061687

Non-Patent Documents

-   Non-patent document 1: American College of Chest Physicians,    CHEST/101/6-/JUNE, 1992:1481-1483-   Non-patent document 2: Koji Suzuki, Igaku no Ayumi (Progress of    Medicine), Vol. 125, 901 (1983)-   Non-patent document 3: K. Gomi et al., Blood, 75, 1396-1399 (1990)-   Non-patent document 4: M. Zushi et al., J. Biol. Chem., 246,    10351-10353 (1989)-   Non-patent document 5: D. Z. Wen et al., Biochemistry, 26, 4350-4357    (1987)-   Non-patent document 6: S. M. Bates et al., Br. J. Pharmacol., 144,    1017-1028 (2005)

SUMMARY OF THE INVENTION Object to be Achieved by the Invention

An object of the present invention is to provide a medicament or methodfor effective therapeutic treatment or improvement of sepsis in a severeseptic patient.

Means for Achieving the Objects

International Normalized Ratio (henceforth also abbreviated as “INR”) ina plasma specimen of a patient with sepsis is known to meancoagulopathy. For example, in Congress of Critical Care Medicine (CCM)held in 2003, INR>1.5 is reported as well as aPPT>60 seconds as criteriaof coagulopathy (Crit. Care Med., 31, pp. 1250-1256 (2003)). However,the value of INR has not yet been authorized as a clear criterion,because the value has not been verified through clinical trials and thelike. Actually, through Phase III clinical trial for treatment ofpatients with severe sepsis, Tifagofin as a tissue factor pathwayinhibitor in a class of anticoagulants is reported to have achieved morefavorable result in a group of patients with INR≦1.2 than a group ofpatients with INR>1.2 as a result of clinical test in which patientswith INR>1.2 were mainly targeted (JAMA, July 9, Vo. 290, No. 2, pp.238-247 (2003)). Whilst as another result of the aforementioned clinicaltest, among the class of patients with INR>1.2, the drug is reported tohave achieved higher effect in patients with INR>1.5 than in patientswith INR>1.2. Further, with Xigris, which is the only drug that has beenverified to be effective against sepsis through clinical study,prolongation of prothrombin-time (PT) was observed in most of thepatients in the study (93.4%).

As explained above, in the therapeutic treatment of patients with sepsisby an anticoagulant, high efficacy is expected by choosing a class ofpatients accompanied with coagulopathy in view of some of the clinicalstudy results. However, it is considered that the definition ofcoagulopathy has not yet been authorized, because, for example, clinicalresults to the contrary were also obtained. In other words, how anexcellent result can be obtained by choosing target patients by means ofthe INR value has not been clarified, and there is no common technicalknowledge that what level of the INR value of a patient with sepsisassures particular effectiveness of the drug. With regard to correlationof the INR value with clinical effectiveness, it is considered that onlya limited part of the correlation has been known as case-by-case basisas for some of individual drugs.

Under the circumstances, the inventors devoted their attention tothrombomodulin among anticoagulants, and conducted various researches ontherapeutic and/or improving effect against sepsis. As a result, theyunexpectedly found that sepsis can be therapeutically treated and/orimproved more effectively in severe septic patients with one or moreorgan dysfunctions (except severe septic patient with organ dysfunctionlimited to the liver or kidney) than in severe septic patients withoutorgan dysfunction when the INR value of the patients is more than 1.4,in other words, as for therapeutic treatment and/or improvement ofsepsis by thrombomodulin, the inventors found that there is a particularcorrelation between severe septic patients with one or more organdysfunctions, among the class of septic patients, and the INR value morethan 1.4, which is unexpected by one of ordinary skill in the art.Further surprisingly, the inventors found that, for severe septicpatients with the INR value more than 1.4 and equal to or less than 1.6,particularly remarkable effect was achieved in that a difference inmortality rate between thrombomodulin group and placebo group was higherthan 15%, and as a result, they accomplished the present invention. Inview of the difference in mortality rate of about 6% between drug groupand placebo group achieved by Xigris (N. Engl. J. Med., 344, No. 10,March 8, pp. 699-709 (2001)), which is a sole commercially availabledrug in Europe for treatment of sepsis, the value of 15% as thedifference in mortality rate is a remarkable value as high as about2.5-fold, and therefore, it can be recognized that one of embodiments ofthe present invention achieves unexpectedly surprising effect.

Specifically, the present invention includes the followings:

[A1] A medicament for therapeutic treatment and/or improvement of sepsiswhich comprises thrombomodulin as an active ingredient, wherein saidmedicament is to administer to a patient with severe sepsis accompaniedwith one or more organ dysfunctions, wherein a value of InternationalNormalized Ratio (INR) of a plasma specimen obtained from said patientis more than 1.4.[A1-2] A medicament for therapeutic treatment and/or improvement ofsepsis accompanied with coagulopathy which comprises thrombomodulin asan active ingredient, wherein said medicament is to administer to apatient with severe sepsis accompanied with one or more organdysfunctions, wherein a value of International Normalized Ratio (INR) ofa plasma specimen obtained from said patient is more than 1.4.[A2] The medicament according to [A1] or [A1-2] mentioned above, whereinsaid medicament is to administer to a patient with severe sepsisaccompanied with one or more organ dysfunctions, wherein a value ofInternational Normalized Ratio (INR) of a plasma specimen obtained fromsaid patient is more than 1.4 and equal to or less than 1.6.[A3] The medicament according to any one of [A1] to [A2] mentionedabove, wherein the patient with severe sepsis is a severe septic patientwho is not a septic patient with organ dysfunction limited to the liveror kidney.

When the referred item numbers are indicated with such a range as “[A1]to [A2]” mentioned above, and the range includes an item indicated witha number having a subnumber such as [A1-2], it is meant that the itemindicated with the number having a subnumber such as [A1-2] is alsocited. The same shall apply to the following definitions.

[A4] The medicament according to any one of [A1] to [A3] mentionedabove, wherein said medicament is to administer to a patient with severesepsis accompanied with one or more organ dysfunctions selected from thegroup consisting of liver dysfunction, kidney dysfunction, respiratoryorgan dysfunction, and circulatory organ dysfunction.[A5] The medicament according to any one of [A1] to [A4] mentionedabove, wherein the thrombomodulin is a soluble thrombomodulin.[A5-2] The medicament according to any one of [A1] to [A5] mentionedabove, wherein the soluble thrombomodulin has the following properties(1) to (4):(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time, and(4) an action of suppressing platelet aggregation caused by thrombin.[A5-3] The medicament according to any one of [A1] to [A5] mentionedabove, wherein the soluble thrombomodulin has the following properties(1) to (5):(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time,(4) an action of suppressing platelet aggregation caused by thrombin,and(5) anti-inflammatory action.[A6] The medicament according to any one of [A1] to [A5-3] mentionedabove, wherein the thrombomodulin is a peptide obtainable from atransformed cell prepared by transfecting a host cell with a DNA codingfor the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.[A7] The medicament according to any one of [A1] to [A6] mentionedabove, wherein the soluble thrombomodulin is a peptide containing theamino acid sequence of (i-1) or (i-2) mentioned below, and said peptideis a peptide having the thrombomodulin activities;(i-1) the amino acid sequence of the positions 19 to 516 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(i-2) the amino acid sequence of (i-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues.[A7-2] The medicament according to any one of [A1] to [A6] mentionedabove, wherein the soluble thrombomodulin is a peptide containing:(i) the amino acid sequence of the positions 367 to 480 in the aminoacid sequence of SEQ ID NO: 9 or 11, and the amino acid sequence of(ii-1) or (ii-2) mentioned below, and said peptide is a peptide havingthe thrombomodulin activities:(ii-1) the amino acid sequence of the positions 19 to 244 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(ii-2) the amino acid sequence of (ii-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues,[A8] The medicament according to any one of [A1] to [A7-2] mentionedabove, wherein the thrombomodulin is administered at a dose of 0.005 to1 mg/kg within 5 minutes by intravenous bolus administration.[B1] A method for therapeutic treatment and/or improvement of sepsis,which comprises the step of administrating thrombomodulin to a patientwith severe sepsis, wherein a value of International Normalized Ratio(INR) of a plasma specimen obtained from said patient is more than 1.4.[B1-2] A method for therapeutic treatment and/or improvement of sepsisaccompanied with coagulopathy, which comprises the step ofadministrating thrombomodulin to a patient with severe sepsis, wherein avalue of International Normalized Ratio (INR) of a plasma specimenobtained from said patient is more than 1.4.[B-2] The method according to [B1] or [B1-2] mentioned above, whichcomprises the step of administrating thrombomodulin to a patient withsevere sepsis accompanied with one or more organ dysfunctions, wherein avalue of International Normalized Ratio (INR) of a plasma specimenobtained from said patient is more than 1.4 and equal to or less than1.6.[B3] The method according to any one of [B1] to [B-2] mentioned above,wherein the patient with severe sepsis is a severe septic patient who isnot a septic patient with organ dysfunction limited to the liver orkidney.[B4] The method according to any one of [B1] to [B3] mentioned above,which comprises the step of administrating thrombomodulin to a patientwith severe sepsis accompanied with one or more organ dysfunctionsselected from the group consisting of liver dysfunction, kidneydysfunction, respiratory organ dysfunction, and circulatory organdysfunction.[B5] The method according to any one of [B1] to [B4] mentioned above,wherein the thrombomodulin is a soluble thrombomodulin.[B6] The method according to any one of [B1] to [B5] mentioned above,wherein the thrombomodulin is a peptide obtainable from a transformedcell prepared by transfecting a host cell with a DNA coding for theamino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 11.[B7] The method according to any one of [B1] to [B6] mentioned above,wherein the soluble thrombomodulin is a peptide containing the aminoacid sequence of (i-1) or (i-2) mentioned below, and said peptide is apeptide having the thrombomodulin activities; (i-1) the amino acidsequence of the positions 19 to 516 in the amino acid sequence of SEQ IDNO: 9 or 11, or(i-2) the amino acid sequence of (i-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues.[B8] The method according to any one of [B1] to [B7] mentioned above,wherein the thrombomodulin is administered at a dose of 0.005 to 1 mg/kgwithin 5 minutes by intravenous bolus administration.[B8-2] The method according to any one of [B1] to [B8] mentioned above,wherein the thrombomodulin is a thrombomodulin having the characteristicfeatures mentioned in [A5-2], [A5-3] or [A7-2] mentioned above.[B9] Use of thrombomodulin as a medicament for therapeutic treatmentand/or improvement of sepsis, wherein said medicament is to administerto a patient with severe sepsis accompanied with one or more organdysfunctions, wherein a value of International Normalized Ratio (INR) ofa plasma specimen obtained from said patient is more than 1.4.[B9-2] The use according to [B9] mentioned above, wherein thethrombomodulin is a thrombomodulin having the characteristic featuresmentioned in [A5-2], [A5-3] or [A7-2] mentioned above.[B9-3] The use according to [B9] mentioned above, which has thecharacteristic features mentioned in any one of [A1] to [A8] mentionedabove.[C1] A medicament for therapeutic treatment and/or improvement ofdisseminated intravascular coagulation, wherein said medicament is toadminister to a patient with disseminated intravascular coagulationaccompanied with one or more organ dysfunctions, wherein a value ofInternational Normalized Ratio (INR) of a plasma specimen obtained fromsaid patient is higher than 1.4.[C2] The medicament according to [C1] mentioned above, which has thecharacteristic features mentioned in any one of [A1] to [A8] mentionedabove.

Effect of the Invention

With the medicament of the present invention containing thrombomodulin,sepsis in a severe septic patient, wherein a value of InternationalNormalized Ratio (INR) of a plasma specimen obtained from said patientis more than 1.4, can be effectively treated and/or improved.

MODES FOR CARRYING OUT THE INVENTION

Hereafter, several preferred embodiments of the present invention(preferred modes for carrying out the invention, henceforth alsoreferred to as “embodiments” in the specification) will be specificallyexplained. However, the scope of the present invention is not limited tothe specific embodiments explained below.

The thrombomodulin of this embodiment preferably is known to have anaction of (1) selectively binding to thrombin (2) to promote activationof Protein C by thrombin. In addition, it is preferred that thethrombomodulin is confirmed to generally have (3) an action of extendingthrombin clotting time, (4) an action of suppressing plateletaggregation caused by thrombin, and/or (5) anti-inflammatory action.Such actions possessed by thrombomodulin may be referred to asthrombomodulin activities.

As the thrombomodulin activities, thrombomodulin preferably has theactions of (1) and (2) mentioned above, and more preferably has theactions of (1) to (4) mentioned above. As the thrombomodulin activities,thrombomodulin more preferably has all of the actions of (1) to (5)mentioned above.

The action of thrombomodulin to bind with thrombin can be confirmed bythe study methods described in various known publications such asThrombosis and Haemostasis, 70(3):418-422 (1993) and The Journal ofBiological Chemistry, 264, 9, pp. 4872-4876 (1989). As for the action ofpromoting activation of Protein C by thrombin, degree of the activity ofpromoting the activation of Protein C by thrombin or presence or absenceof the action can be easily confirmed by the study methods clearlydescribed in various known publications including, for example, JapanesePatent Unexamined Publication No. 64-6219. Further, the action ofextending thrombin clotting time, and/or the action of suppressingplatelet aggregation caused by thrombin can be similarly and easilyconfirmed. Furthermore, the anti-inflammatory action can also beconfirmed by the study methods described in various known publicationsincluding, for example, Blood, 112:3361-3670 (2008) and The Journal ofClinical Investigation, 115, 5:1267-1274 (2005).

The thrombomodulin used for the present invention is not particularlylimited so far as having the thrombomodulin activities. Thethrombomodulin is preferably a soluble thrombomodulin under thecondition without surfactants. The solubility of the solublethrombomodulin in water such as distilled water used for injection (inthe absence of a surfactant such as Triton X-100 or polidocanol, andgenerally around the neutral pH range) is preferably, for example, 1mg/mL or more or 10 mg/mL or more; preferably 15 mg/mL or more or 17mg/mL or more; more preferably 20 mg/mL or more, 25 mg/mL or more, or 30mg/mL or more; particularly preferably 60 mg/mL or more. In some cases,the solubility is, for example, 80 mg/mL or more, or 100 mg/mL or more.For determining whether or not a soluble thrombomodulin is successfullydissolved in water, it is understood that clear appearance of a solutionand the absence of apparently observable insoluble substances is servedas simple criteria, after the soluble thrombomodulin is dissolved inwater and the solution is observed by visual inspection, for example,just under a white light at a position corresponding to an illuminationof approximately 1000 luxes. It is also possible to observe the presenceor absence of any residue after filtration.

The molecular weight of the thrombomodulin is not limited so far that ithas the thrombomodulin activities as described above. The molecularweight is preferably 100,000 or smaller, more preferably 90,000 orsmaller, still more preferably 80,000 or smaller, most preferably 70,000or smaller, and the molecular weight is preferably 50,000 or larger,most preferably 60,000 or larger. The molecular weight of the solublethrombomodulin can be easily measured by ordinary methods for measuringmolecular weight of protein. Measurement by mass spectrometry ispreferred, and MALDI-TOF-MS method is more preferred. For obtainingsoluble thrombomodulin having a molecular weight within a desired range,a soluble thrombomodulin, which is obtained by culturing a transformantcell prepared by transfecting a host cell with a DNA encoding solublethrombomodulin using a vector, can be subjected to fractionation usingcolumn chromatography or the like as described later.

The thrombomodulin used for the present invention preferably comprisesthe amino acid sequence consisting of the amino acid residues at thepositions 19 to 132 of SEQ ID NO: 1, which has been known as the centralportion of the thrombomodulin activities of human thrombomodulin, andthe thrombomodulin is not particularly limited, so long as thethrombomodulin comprises the amino acid sequence consisting of the aminoacid residues at the positions 19 to 132 of SEQ ID NO: 1. The amino acidsequence consisting of the amino acid residues at the positions 19 to132 of SEQ ID NO: 1 may be naturally or artificially mutated, so long asthe sequence has an action to promote the activation of Protein C bythrombin, namely, one of the thrombomodulin activities. Specifically,the sequence may comprise substitution, deletion, or addition of one ormore amino acid residue in the amino acid sequence consisting of theamino acid residues at the positions 19 to 132 of SEQ ID NO: 1.Acceptable level of the mutation is not particularly limited, so long asthe amino acid sequence has the thrombomodulin activities. An exampleincludes a homology 50% or more as amino acid sequences, and thehomology is preferably 70% or more, more preferably 80% or more, furtherpreferably 90% or more, particularly preferably 95% or more, and mostpreferably 98% or more. Such mutated amino acid sequence includingsubstitution, deletion or addition of one or more amino acid residues isreferred to as homologous mutation sequence. As described later, thesemutated amino acid sequences can be easily produced by using ordinarygene manipulation techniques. The thrombomodulin is not particularlylimited so far that it has the aforementioned sequence and the action ofselectively binding to thrombin to promote activation of Protein C bythrombin at least as the whole thrombomodulin, but the thrombomodulinpreferably also has the anti-inflammatory action.

The amino acid sequence of SEQ ID NO: 3 comprises the mutation of Val asthe amino acid at the position 125 of the sequence of SEQ ID NO: 1 toAla. The thrombomodulin used for the present invention also preferablycomprises the amino acid sequence from the position 19 to 132 of SEQ IDNO: 3.

As described above, although the thrombomodulin used for the presentinvention is not particularly limited so long that the thrombomodulinhas at least the amino acid sequence from the position 19 to 132 of SEQID NO: 1 or 3, or a homologous mutation sequence thereof, and comprisesat least a peptide sequence having the thrombomodulin activities,preferred examples of the thrombomodulin include a peptide consisting ofthe sequence from the position 19 to 132 or 17 to 132 in either of SEQID NO: 1 or SEQ ID NO: 3, and a peptide consisting of a homologousmutation sequence of the aforementioned sequence and having at least thethrombomodulin activities. A peptide consisting of the sequence from theposition 19 to 132 in either of SEQ ID NO: 1 or SEQ ID NO: 3 is morepreferred. In another embodiment, a peptide consisting of a homologousmutation sequence of the sequence from the position 19 to 132 or 17 to132 in either of SEQ ID NO: 1 or SEQ ID NO: 3 and having at least thethrombomodulin activities is more preferred.

As another embodiment of the thrombomodulin according to the presentinvention, the thrombomodulin preferably comprises the amino acidsequence from the positions 19 to 480 of SEQ ID NO: 5, which is notparticularly limited so long as the thrombomodulin comprises the aminoacid sequence from the position 19 to 480 of SEQ ID NO: 5. The aminoacid sequence from the positions 19 to 480 of SEQ ID NO: 5 may be ahomologous mutation sequence thereof, so long as the sequence has anaction to promote the activation of Protein C by thrombin, i.e., one ofthe thrombomodulin activities.

The sequence of SEQ ID NO: 7 comprises the mutation of Val as the aminoacid at the position 473 of the sequence of SEQ ID NO: 5 to Ala. Thethrombomodulin used for the present invention also preferably comprisesthe amino acid sequence from the position 19 to 480 of SEQ ID NO: 7.

As described above, although the thrombomodulin used for the presentinvention is not particularly limited so long as the thrombomodulin hasat least the sequence from the position 19 to 480 in either of SEQ IDNO: 5 or SEQ ID NO: 7, or a homologous mutation sequence thereof, andcomprises at least a peptide sequence having the thrombomodulinactivities, preferred examples of the thrombomodulin include a peptideconsisting of the sequence from the position 19 to 480 or 17 to 480 ineither of SEQ ID NO: 5 or SEQ ID NO: 7, and a peptide consisting of ahomologous mutation sequence of the aforementioned sequence and havingat least the thrombomodulin activities. A peptide consisting of thesequence from the position 19 to 480 of SEQ ID NO: 5 or 7 is morepreferred. In another embodiment, a peptide consisting of a homologousmutation sequence of the sequence from the position 19 to 480 or 17 to480 in either of SEQ ID NO: 5 or SEQ ID NO: 7, and having thethrombomodulin activities is more preferred.

As another embodiment of the thrombomodulin according to the presentinvention, the thrombomodulin preferably comprises the amino acidsequence from the position 19 to 515 of SEQ ID NO: 9, which is notparticularly limited so long as the thrombomodulin comprises the aminoacid sequence from the position 19 to 515 of SEQ ID NO: 9. The aminoacid sequence from the position 19 to 515 of SEQ ID NO: 9 may be ahomologous mutation sequence thereof, so long as the sequence has anaction to promote the activation of Protein C by thrombin, i.e., thethrombomodulin activities.

The amino acid sequence of SEQ ID NO: 11 comprises the mutation of Valas the amino acid at the position 473 of SEQ ID NO: 9 to Ala. Thethrombomodulin used for the present invention also preferably comprisesthe amino acid sequence from the position 19 to 515 of SEQ ID NO: 11.

As described above, although the thrombomodulin used for the presentinvention is not particularly limited so long as the thrombomodulin hasat least the sequence from the position 19 to 515 in either of SEQ IDNO: 9 or SEQ ID NO: 11, or a homologous mutation sequence thereof, andcomprises a peptide sequence having at least the thrombomodulinactivities, more preferred examples include a peptide having thesequence from position 19 to 516, 19 to 515, 17 to 516, or 17 to 515 ineither of SEQ ID NO: 9 or SEQ ID NO: 11, and a peptide consisting of ahomologous mutation sequence of the aforementioned sequence and havingat least the thrombomodulin activities. A peptide having the sequencefrom the position 19 to 516, 19 to 515, 17 to 516, or 17 to 515 of SEQID NO: 9 is particularly preferred. A mixture thereof is also apreferred example. In another embodiment, a peptide having the sequencefrom the position 19 to 516, 19 to 515, 17 to 516, or 17 to 515 of SEQID NO: 11 is particularly preferred. A mixture thereof is also apreferred example. Further, a peptide consisting of a homologousmutation sequence thereof and having at least the thrombomodulinactivities is also a preferred example. It is preferred that the solublethrombomodulin also has the anti-inflammatory action.

A peptide having a homologous mutation sequence is as described above,and means a peptide that may comprise substitution, deletion, oraddition of at least one, namely, one or more, preferably several (forexample, 1 to 20, preferably 1 to 10, more preferably 1 to 5,particularly preferably 1 to 3) amino acid residues, in the amino acidsequence of the subjected peptide. Although acceptable level of mutationis not particularly limited so long as the peptide has thethrombomodulin activities, an example of the acceptable level ofhomology includes 50% or more as an amino acid sequences, and thehomology may be preferably 70% or more, more preferably 80% or more,further preferably 90% or more, particularly preferably 95% or more, andmost preferably 98% or more.

Preferred examples of the thrombomodulin used for the present inventionalso include the peptide consisting of the sequence of SEQ ID NO: 14(462 amino acid residues), the peptide consisting of the sequence of SEQID NO: 8 (272 amino acid residues), and the peptide consisting of thesequence of SEQ ID NO: 6 (236 amino acid residues) described in JapanesePatent Unexamined Publication No. 64-6219.

The thrombomodulin used for the present invention is not particularlylimited so long as the thrombomodulin has at least the amino acidsequence from the position 19 to 132 in either of SEQ ID NO: 1 or SEQ IDNO: 3. As such a thrombomodulin, a peptide having at least the aminoacid sequence from the position 19 to 480 in either of SEQ ID NO: 5 orSEQ ID NO: 7 is preferred, and a peptide having at least the amino acidsequence from the position 19 to 515 in either of SEQ ID NO: 9 or SEQ IDNO: 11 is more preferred. A more preferred example of the peptide havingat least the amino acid sequence from the position 19 to 515 in eitherof SEQ ID NO: 9 or SEQ ID NO: 11 is a peptide having the sequence fromthe position 19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or19 to 514 in either of SEQ ID NO: 9 or SEQ ID NO: 11. Furthermore, amixture of peptides each consisting of the sequence from the position 19to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or 19 to 514 ineither of SEQ ID NO: 9 or SEQ ID NO: 11 is also a preferred example.

In the case of the aforementioned mixture, the mixing ratio of a peptidethat starts from the position 17 and a peptide that starts from theposition 19 for each of SEQ ID NOS: 9 and 11 is, for example, 30:70 to50:50, preferably 35:65 to 45:55.

Further, the mixing ratio of a peptide that terminates at the position514, a peptide that terminates at the position 515, and a peptide thatterminates at the position 516 for each of SEQ ID NOS: 9 and 11 is, forexample, 0:0:100 to 0:90:10, or 0:70:30 to 10:90:0, or 10:0:90 to20:10:70, if desired.

The mixing ratio of the peptides can be determined by an ordinarymethod.

The sequence of the positions 19 to 132 in SEQ ID NO: 1 corresponds tothe sequence of the positions 367 to 480 in SEQ ID NO: 9, and thesequence of the positions 19 to 480 in SEQ ID NO: 5 corresponds to thesequence of the positions 19 to 480 in SEQ ID NO: 9. Further, thesequence of the positions 19 to 132 in SEQ ID NO: 3 corresponds to thesequence of the positions 367 to 480 in SEQ ID NO: 11, and the sequenceof the positions 19 to 480 in SEQ ID NO: 7 corresponds to the sequenceof the positions 19 to 480 in SEQ ID NO: 11. Furthermore, all thesequences of the positions 1 to 18 in SEQ ID NOS: 1, 3, 5, 7, 9 and 11are identical sequences.

As described below, these thrombomodulins according to the presentinvention can be obtained from transformant cells prepared bytransfecting host cells with a DNA encoding the peptide (specifically,the nucleotide sequences of SEQ ID NOS: 2, 4, 6, 8, 10, 12, and thelike) by using a vector.

It is sufficient that these peptides only have the aforementioned aminoacid sequences, and a sugar chain may be attached or not attached, whichnot particularly limited. In gene manipulation techniques, a type of asugar chain, a position to which a sugar chain is added, and a level ofaddition thereof differ depending on a type of host cells used, and anytechniques may be used. As for binding position of a sugar chain and atype thereof, facts described in Japanese Patent Unexamined PublicationNo. 11-341990 are known, and the thrombomodulins according to thepresent invention may be added with the same sugar chain at the sameposition. Two types of N-linked sugar chains, those of fucosylbiantennary type and fucosyl triantennary type, may bind to thethrombomodulin of this embodiment, and ratio thereof is, for example,100:0 to 60:40, preferably 95:5 to 60:40, more preferably 90:10 to70:30. The ratio of these sugar chains can be measured on atwo-dimensional sugar chain map described in Biochemical ExperimentalMethods, Vol. 23, Methods of Researches on Glycoprotein Sugar Chains,Japan Scientific Societies Press (1990), and the like. Furthermore, whena sugar composition of the thrombomodulin of this embodiment isexamined, neutral saccharides, aminosaccharides, and sialic acid aredetected, of which content may be, each independently for example, 1 to30%, preferably 2 to 20%, more preferably 5 to 10%, in terms of weightratio based on the protein content. The sugar contents can be measuredby the methods described in Lecture of New Biochemical Experiments, Vol.3, Sugar I, Glycoprotein (Book 1), Tokyo Kagaku Dojin (1990) (neutralsaccharides: phenol-sulfuric acid method, aminosaccharides: Elson-Morganmethod, sialic acid:periodic acid-resorcinol method).

Although the method for obtaining thrombomodulin is not limited toobtaining it by genetic manipulation as described later, as a signalsequence that can be used for expression where the thrombomodulin isobtained by gene manipulation, a nucleotide sequence encoding the aminoacid sequence of the positions 1 to 18 in SEQ ID NO: 9, and a nucleotidesequence encoding the amino acid sequence of the positions 1 to 16 inSEQ ID NO: 9 can be used, and other known signal sequences such as thesignal sequence of human tissue plasminogen activator can also be used(International Publication WO88/9811).

When a DNA sequence encoding thrombomodulin is introduced into a hostcell, examples of preferred methods include a method of incorporating aDNA sequence encoding thrombomodulin into, preferably, a vector, morepreferably an expression vector capable of being expressed in animalcells, and then introducing the DNA with the vector. An expressionvector is a DNA molecule that is constituted with a promoter sequence, asequence for adding a ribosome binding site to mRNA, a DNA sequenceencoding a protein to be expressed, a splicing signal, a terminatorsequence for transcription termination, a replication origin sequence,and the like. Examples of preferred animal cell expression vectorinclude pSV2-X reported by Mulligan R. C. et al. (Proc. Natl. Acad. Sci.U.S.A., 78, 2072 (1981)); pBP69T (69-6) reported by Howley P. M. et al.(Methods in Emzymology, 101, 387 (1983), Academic Press), and the like.Further, there is also another preferred embodiment in which DNA isintroduced into an expression vector expressible in a microorganism.

Examples of host cell that can be used in production of such peptides asmentioned above include animal cells. Examples of the animal cellsinclude Chinese hamster ovary (CHO) cells, COS-1 cells, COS-7 cells,VERO (ATCC CCL-81) cells, BHK cells, canine kidney-derived MDCK cells,hamster AV-12-664 cells, and the like. In addition, examples of hostcell derived from human include HeLa cells, WI38 cells, human 293 cells,and PER.C6 cells. Of these cells, CHO cells are very common andpreferred, and among the CHO cells, dihydrofolate reductase(DHFR)-deficient CHO cells are more preferred.

In a gene manipulation process or a peptide production process,microorganisms such as Escherichia coli are also often used. Ahost-vector system suitable for each process is preferably used, and anappropriate vector system can also be selected for the aforementionedhost cells. A thrombomodulin gene used in a genetic recombinationtechnique has been cloned. Examples of production of thrombomodulin bysuch a gene recombination technique have been disclosed, and further,methods for purifying thrombomodulin to obtain a purified productthereof are also known (Japanese Patent Unexamined Publication Nos.64-6219, 2-255699, 5-213998, 5-310787, 7-155176; and J. Biol. Chem.,26410351-10353 (1989)). Therefore, the thrombomodulin used for thepresent invention can be produced by using the methods described in theaforementioned reports, or by similar methods. For example, JapanesePatent Unexamined Publication No. 64-6219 discloses the Escherichia coliK-12 strain DH5 (ATCC Accession No. 67283) containing a plasmid pSV2TMJ2that contains a DNA encoding the full-length thrombomodulin. This strainre-deposited at the former National Institute of Bioscience andHuman-Technology (currently Independent Administrative Institution,National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary) (Escherichia coliDH5/pSV2TMJ2) (FERM BP-5570) can also be used. The thrombomodulinaccording to the present invention can be prepared by a known genemanipulation technique using a DNA encoding the full-lengththrombomodulin as a starting material.

The thrombomodulin of this embodiment may be prepared by aconventionally known method or a similar method. For example, theaforementioned method of Yamamoto et al. (Japanese Patent UnexaminedPublication No. 64-6219) or the method described in Japanese PatentUnexamined Publication No. 5-213998 can be referred to. Specifically,for example, a DNA encoding the amino acid sequence of SEQ ID NO: 9 isprepared from a human-derived thrombomodulin gene by a gene manipulationtechnique, and may be further modified as required. For suchmodification, in order to obtain a DNA encoding the amino acid sequenceof SEQ ID NO: 11 (which specifically consists of the nucleotide sequenceof SEQ ID NO: 12), codons encoding the amino acid at the position 473 inthe amino acid sequence of SEQ ID NO: 9 (in particular, the nucleotideat the position 1418 in SEQ ID NO: 10) are mutated by site-directedmutagenesis according to the method described by Zoller M. J. et al.(Method in Enzymology, 100:468-500 (1983), Academic Press). For example,by using a synthetic DNA for mutation having the nucleotide sequence ofSEQ ID NO: 13, the nucleotide T at the position 1418 in SEQ ID NO: 10may be converted to the nucleotide C to obtain a mutated DNA.

The DNA prepared as described above is incorporated into, for example,Chinese hamster ovary (CHO) cells to obtain transformant cells. Suchcells are subjected to appropriate selection, and thrombomodulinpurified by a known method can be produced from a culture solutionobtained by culturing a selected cell. As described above, the DNA (SEQID NO: 10) encoding the amino acid sequence of SEQ ID NO: 9 ispreferably transfected into the aforementioned host cell.

The method for producing thrombomodulin of this embodiment is notlimited to the aforementioned method. For example, it is also possibleto extract and purify the thrombomodulin from urine, blood, other bodyfluids and the like, or extract and purify the thrombomodulin from atissue producing thrombomodulin or a culture of the aforementionedtissue and the like. Further, the thrombomodulin may be furthersubjected to a cleavage treatment using a protease, as required.

For the culture of the aforementioned transformant cell, a medium usedfor ordinary cell culture may be used, and it is preferable to culturethe transformant cell in various kinds of media in advance to choose anoptimal medium. For example, a known medium such as MEM medium, DMEMmedium, and 199 medium may be used as a base medium, and a furtherimproved medium or a medium added with supplements for various media maybe used. Examples of the culture method include serum culture, in whichculture is performed in a medium containing blood serum, and serum-freeculture, in which culture is performed in a medium not containing bloodserum. Although the culture method is not particularly limited, theserum-free culture is preferred.

When serum is added to a medium in the case of the serum culture, bovineserum is preferred. Examples of bovine serum include fetal bovine serum,neonate bovine serum, calf bovine serum, adult bovine serum, and thelike, and any of these examples may be used so far that the serum issuitable for the cell culture. As the serum-free medium used in theserum-free culture, commercially available media can be used. Serum-freemedia suitable for various cells are marketed, and for example, for theCHO cell, CD-CHO, CHO-S-SFMII and CHO-III-PFM are sold by Invitrogen,and IS CHO, IS CHO-CD medium, and the like are sold by IrvineScientific. These media may be used without any treatment, or they maybe improved or added with supplements and used. Examples of theserum-free medium further include the DMEM medium containing 5 mg/L eachof insulin, transferrin, and selenious acid. As described above, themedium is not particularly limited so far that the medium can be used toproduce the thrombomodulin of this embodiment. The culture method is notparticularly limited, and any of batch culture, repetitive batchculture, fed-batch culture, perfusion culture, and the like may be used.

When the thrombomodulin used for the present invention is prepared bythe aforementioned cell culture method, diversity may be observed in theN-terminus amino acid due to posttranslational modification of theprotein. For example, the amino acid of the position 17, 18, 19 or 22 inSEQ ID NO: 9 may serve as the N-terminus amino acid. Further, forexample, the N-terminus amino acid may be modified so that the glutamicacid at the position 22 is changed to pyroglutamic acid. It is preferredthat the amino acid of the position 17 or 19 serves as the N-terminusamino acid, and it is more preferred that the amino acid of the position19 serves as the N-terminus amino acid. Further, there is also anotherembodiment in which the amino acid of the position 17 serves as theN-terminus amino acid, which is a preferred embodiment. As for themodification, diversity and the like mentioned above, similar examplescan be mentioned for the sequence of SEQ ID NO: 11.

Further, when the soluble thrombomodulin is prepared by using a DNAhaving the nucleotide sequence of SEQ ID NO: 10, diversity of theC-terminus amino acid may be observed, and a peptide shorter by oneamino acid residue may be produced. Specifically, the C-terminus aminoacid may be modified so that the amino acid of the position 515 servesas the C-terminus amino acid, and further the position 515 is amidated.Further, a peptide shorter by two amino acid residues may be produced.Specifically, the amino acid of the position 514 may serve as theC-terminus amino acid. Therefore, any of peptides having significantdiversity of the N-terminus amino acid and C-terminus amino acid, or amixture of them may be produced. It is preferred that the amino acid ofthe position 515 or the amino acid of the position 516 serves as theC-terminus amino acid, and it is more preferred that the amino acid ofthe position 516 serves as the C-terminus amino acid. Further, there isalso another embodiment in which the amino acid of the position 514serves as the C-terminus amino acid, which is a preferred embodiment.Concerning the modification, diversity and the like described above, thesame shall apply to a DNA having the nucleotide sequence of SEQ ID NO:12.

The thrombomodulin obtained by the method described above may be amixture of peptides having diversity in the N-terminus and C-terminusamino acids. Specific examples include a mixture of peptides having thesequences of the positions 19 to 516, positions 19 to 515, positions 19to 514, positions 17 to 516, positions 17 to 515, and positions 17 to514 in SEQ ID NO: 9.

Then, isolation and purification of thrombomodulin from a culturesupernatant or culture obtained as described above can be carried out byknown methods [edited by Takeichi Horio, Tanpakushitsu/Koso no KisoJikken Ho (Fundamental Experimental Methods for Proteins and Enzymes)(1981)]. For example, it is preferable to use ion exchangechromatography or adsorption chromatography, which utilizes aninteraction between thrombomodulin and a chromatographic carrier onwhich functional groups having a charge opposite to that ofthrombomodulin are immobilized. Another preferred example is affinitychromatography utilizing specific affinity with thrombomodulin.Preferred examples of adsorbent include thrombin that is a ligand ofthrombomodulin and an anti-thrombomodulin antibody. As the antibody,anti-thrombomodulin antibodies having appropriate properties orrecognizing appropriate epitopes can be used. Examples include, forexample, those described in Japanese Patent Publication (Kokoku) No.5-42920, Japanese Patent Unexamined Publication Nos. 64-45398 and6-205692 and the like. Other examples include gel filtrationchromatography and ultrafiltration, which utilize the molecular size ofthrombomodulin. Other examples further include hydrophobicchromatography that utilizes hydrophobic bond between a chromatographiccarrier on which hydrophobic groups are immobilized, and a hydrophobicportion of thrombomodulin. Furthermore, hydroxyapatite may be used as acarrier in adsorption chromatography, of which examples include, forexample, those described in Japanese Patent Unexamined Publication No.9-110900. These means may be used in combination, as required. Althoughdegree of purification can be selected depending on a purpose of use andthe like, it is desirable to purify thrombomodulin until a single bandis obtained as a result of electrophoresis, preferably SDS-PAGE, or asingle peak is obtained as a result of gel filtration HPLC or reversephase HPLC of the isolated and purified product. It should be understoodthat, when two or more types of thrombomodulins are used, it ispreferred that only the bands of the thrombomodulins are substantiallyobtained, and it is not required to obtain one single band.

Specific examples of the purification method used in the presentinvention include a purification method using the thrombomodulinactivities as a criterion, for example, a purification method comprisingroughly purifying a culture supernatant or a culture product with an ionexchange column Q-Sepharose Fast Flow to collect a fraction having thethrombomodulin activities; then purifying the fraction with an affinitycolumn, DIP-thrombin-agarose (diisopropylphosphorylthrombin agarose)column, as the main purification step to recover a fraction havingpotent thrombomodulin activities; then concentrating the recoveredfraction and followed by gel filtration to obtain a thrombomodulinactive fraction as a purified product (Gomi K. et al., Blood, 75:1396-1399 (1990)). An example of the thrombomodulin activities used asthe criterion is an activity of promoting the activation of Protein C bythrombin. Other preferred examples of the purification method will beexemplified below.

An appropriate ion exchange resin having good adsorptive condition forthrombomodulin is selected and purification by ion exchangechromatography is performed. A particularly preferred example is amethod comprising the use of Q-Sepharose Fast Flow equilibrated with a0.02 mol/L Tris-HCl buffer (pH 7.4) containing 0.18 mol/L NaCl. Afterwashing as required, elution can be performed with a 0.02 mol/L Tris-HClbuffer (pH 7.4) containing 0.3 mol/L NaCl, for example, to obtainthrombomodulin as a roughly purified product.

Then, for example, a substance having specific affinity tothrombomodulin can be immobilized on a resin to perform purification byaffinity chromatography. Preferred examples include aDIP-thrombin-agarose column and an anti-thrombomodulin monoclonalantibody column. In the case of the DIP-thrombin-agarose column, thecolumn is equilibrated beforehand with a 20 mmol/L Tris-HCl buffer (pH7.4) containing 100 mmol/L NaCl and 0.5 mmol/L calcium chloride, and theaforementioned roughly purified product is then charged on the column,washed as required, and then eluted with, for example, a 20 mmol/LTris-HCl buffer (pH 7.4) containing 1.0 mol/L NaCl and 0.5 mmol/Lcalcium chloride to obtain thrombomodulin as a purified product. In thecase of the anti-thrombomodulin monoclonal antibody column, an exampleof the method comprises: contacting an anti-thrombomodulin monoclonalantibody solution in a 0.1 mol/L NaHCO₃ buffer (pH 8.3) containing 0.5mol/L NaCl with Sepharose 4FF (GE Health Care Biosciences) activatedwith CNBr beforehand to obtain the resin Sepharose 4FF coupled with theanti-thrombomodulin monoclonal antibodies, equilibrating the resinfilled in a column beforehand with, for example, a 20 mmol/L phosphatebuffer (pH 7.3) containing 0.3 mol/L NaCl, washing as required, and thenperforming elution with a 100 mmol/L glycine-HCl buffer (pH 3.0)containing 0.3 mol/L NaCl. An effluent may be neutralized with anappropriate buffer to obtain a product as a purified product.

Subsequently, the purified product is adjusted to pH 3.5, and thencharged on a cation exchanger, preferably SP-Sepharose FF (GE HealthCare Biosciences) as a strong cation exchanger, equilibrated with a 100mmol/L glycine-HCl buffer (pH 3.5) containing 0.3 mol/L NaCl, andwashing is performed with the same buffer to obtain a non-adsorptivefraction. The resulting fraction is neutralized with an appropriatebuffer to obtain a highly purified product. These products arepreferably concentrated by ultrafiltration.

Further, it is also preferable to exchange the buffer by gel filtration.For example, a highly purified product concentrated by ultrafiltrationcan be charged on a Sephacryl S-300 column or S-200 column equilibratedwith a 20 mmol/L phosphate buffer (pH 7.3) containing 50 mmol/L NaCl,and then developed for fractionation with a 20 mmol/L phosphate buffer(pH 7.3) containing 50 mmol/L NaCl. The activity for promoting theactivation of Protein C by thrombin can be confirmed to collect anactive fraction and thereby obtain a buffer-exchanged highly purifiedproduct. In order to improve safety, a highly purified product obtainedas described above is preferably filtered through an appropriate filterfor eliminating viruses such as Planova 15N (Asahi Kasei Medical Co.,Ltd.), and then the resultant can be concentrated by ultrafiltration toa desired concentration. Finally, the product is preferably filteredthrough an aseptic filtration filter.

According to the present invention, there is provided a medicament fortherapeutic treatment and/or improvement of sepsis which comprisesthrombomodulin as an active ingredient, wherein said medicament is toadminister to a patient with severe sepsis accompanied with one or moreorgan dysfunctions, wherein a value of International Normalized Ratio(INR) of a plasma specimen obtained from said patient is more than 1.4.

That is, the medicament for therapeutic treatment and/or improvement ofsepsis according to this embodiment is a medicament for therapeutictreatment and/or improvement of sepsis in a patient with severe sepsisaccompanied with one or more organ dysfunctions, wherein a value ofInternational Normalized Ratio (INR) of a plasma specimen obtained fromsaid patient is more than 1.4.

There is also provided a medicament for decreasing mortality of a humanpatient with severe sepsis accompanied with one or more organdysfunctions, wherein a value of International Normalized Ratio (INR) ofa plasma specimen obtained from said patient is more than 1.4.

As for the therapeutic treatment and/or improvement of sepsis, examplesof preferred effects thereof include, for example, “prevention of deathof a patient from sepsis”. Examples also include “prevention ofaggravation of general conditions of a patient by sepsis”.

The sepsis referred to in this embodiment is known as a severe systemicinfectious disease wherein microorganisms continuously or intermittentlyinvade into blood from an infection focus, which disease is induced by adisease such as infectious diseases, malignant tumors, hepaticcirrhosis, renal failure, diabetes, and dystocia, or a therapeutictreatment for injury or disease such as use of indwelling catheter,infusion device, dialysis, and the like and tracheostomy. If thesymptoms advance, a systemic shock is induced by septic shock, i.e.,rapid decrease of blood pressure and peripheral circulatory failure, andlethality is provided by organ dysfunctions of vital organs, such aslung, kidney, liver, heart, alimentary canal, and central nervoussystem. As a complication accompanying sepsis, there is induced adultrespiratory distress syndrome (ARDS) characterized by edema of lungstroma, hemorrhage and acute respiratory failure due to lung capillaryobstruction associated with DIC or activation of neutrophiles andmigration and accumulation thereof in lung parenchyma, which results inextremely bad prognosis.

The sepsis referred to in this embodiment is the systemic inflammatoryresponse syndrome (SIRS) induced by infection. More specifically, itincludes a pathological condition that meets, in addition to thepresence of infection, two or more of the SIRS items ((1) bodytemperature >38° C. or <36° C., (2) heart rate >90/minute, (3)respiration rate >20/minute, or PaCO₂<32 torr, and (4) leucocytecount >12,000/μL or <4000/μL, or bademia >10%), and sepsis can bebasically diagnosed on the basis of such a pathological condition.

There are several methods for diagnosing sepsis, and they are summarizedin Levy M. et al., Crit. Care. Med., 31:1250-1256. For example, thereare a method based on diagnosis performed by a medical practitioner, anda method of using test values and the like Examples of the latterinclude a method in which when two items are fulfilled among the fouritems of (1) body temperature >38° C. or <36° C., (2) heartrate >90/minute, (3) respiration rate >20/minute, or necessity ofartificial respiration, and (4) leucocyte count >12,000/μL or <4000/μL,or bandemia >10%, diagnosis of SIRS is established, and SIRS for which amicroorganism is identified or suspected as the cause thereof isdiagnosed as sepsis [LaRosa S., the homepage of The Cleveland Clinic].Another method similar to the above method is described in Members ofthe American College of Chest Physicians/Society of Critical CareMedicine Consensus Conference: Crit. Care Med., 20, 864-874 (1992).

Examples of the symptoms of sepsis include, for example, bacteriemia,septicemia, systemic inflammatory response syndrome (SIRS), sepsis (SIRSfor which a microorganism is identified or suspected as the causethereof), severe sepsis, septic shock, intractable septic shock, andmultiple organ dysfunction syndrome (henceforth also referred to asMODS) (Harrison's Principles of Internal Medicine, 15th edition oforiginal work, Section 124, pp. 828-833, Medical Science International,Ltd.). The aforementioned conditions are exemplified as symptoms onwhich the medicament of the present invention for therapeutic treatmentand/or improvement is effective.

Although the sepsis is not particularly limited so long as a disease isdiagnosed as sepsis on the basis of the aforementioned diagnosiscriteria, it is preferably sepsis accompanied by abnormal coagulation(sepsis with coagulopathy). Although the coagulopathy is notparticularly limited so long as INR of a plasma specimen obtained frompatient is more than 1.2, it is preferably more than 1.3, morepreferably more than 1.4.

Examples of the bacteriemia include a condition that presence ofbacteria in blood is verified by a positive result of blood culture.

Examples of the septicemia include a condition that presence ofmicroorganisms or other toxins in blood is confirmed.

Examples of the systemic inflammatory response syndrome (SIRS) include acondition of a preliminary stage of DIC, as described above.

Examples of the severe sepsis include sepsis accompanied by one or moresymptoms including organ dysfunction such as metabolic acidosis, organhypoperfusion, acute encephalopathy, oliguria, hypoxemia or disseminatedintravascular coagulation, and hypotension. As sepsis, one presentingorgan dysfunction, organ hypoperfusion, or hypotension is called severesepsis. The organ hypoperfusion or abnormal perfusion includes lacticacidosis, oliguria, mental clouding, and the like. Among the severesepsis, a condition persistently presenting hypotension despite ofsufficient load of fluid therapy is called as septic shock.

More specifically, the severe sepsis referred to in this embodiment isas follows.

Examples of the septic shock include a condition with hypotension (bloodpressure of 90 mmHg or lower or lower than usual blood pressure by 40mmHg or more), not responding to resuscitation by fluid replacement, andaccompanied by organ failure.

Examples of the intractable septic shock include a condition with septicshock continuing over 1 hour or longer, and not responding to ahypertensor with fluid therapy.

Examples of the multiple organ dysfunction syndrome (MODS) include acondition with malfunction of one or more organs, and requiring medicalintervention for maintaining homeostasis.

INR referred to in this embodiment is an examination criterion thatdefines blood coagulopathy. INR means a prothrombin time (henceforthalso abbreviated as PT) normalized as for differences betweenmanufacturing lots of thromboplastin preparations. INR is generallydefined as follows:

INR value=(Coagulation time (sec) of test specimen/Coagulation time(sec) of control specimen)^((ISI value))

In the equation, coagulation time (sec) of test specimen represents PTof test plasma specimen of a subject to be examined, and ISI representsInternational Sensitivity Index.

Examples of the severe sepsis referred to in this embodiment includesepsis accompanied by one or more symptoms including organ dysfunctionsuch as metabolic acidosis, acute encephalopathy, oliguria, hypoxemia ordisseminated intravascular coagulation, and hypotension, as describedabove. The term severe means that the disease is in a critical conditionfor life support. Examples of the severe sepsis include, in particular,sepsis accompanied by one or more organ dysfunctions. Although the organdysfunction is not particularly limited so far that the organdysfunction is induced by sepsis, the organ dysfunction preferablyincludes failure of an organ that is essential for supporting life.Examples of the one or more organ dysfunctions include one or more organdysfunctions selected from the group consisting of circulatory organdysfunction, respiratory organ dysfunction, kidney dysfunction and liverdysfunction, preferred examples include one or more organ dysfunctionsselected from the group consisting of respiratory organ dysfunction,circulatory organ dysfunction, and kidney dysfunction, and morepreferred examples include one or more organ dysfunctions selected fromthe group consisting of respiratory organ dysfunction, and circulatoryorgan dysfunction. Although number of the organ dysfunctions is notparticularly limited so far that the number is one or more, the numbermay be preferably two or more. In particular, it is preferred that thereare two kinds of organ dysfunctions of respiratory organ dysfunction andcirculatory organ dysfunction.

The circulatory organ dysfunction is not particularly limited so long asa generally known circulatory organ dysfunction, and examples include,for example, blood pressure decrease and shock.

The respiratory organ dysfunction is not particularly limited so long asa generally known respiratory organ dysfunction, and examples include,for example, hypoxemia, acute lung injury and dyspnea.

The kidney dysfunction is not particularly limited so long as agenerally known kidney dysfunction, and examples include, for example,renal function disorder, oliguria, and renal failure.

The liver dysfunction is not particularly limited so long as a generallyknows liver dysfunction, and examples include, for example, hepaticfunction disorder, jaundice, hepatic failure, and the like.

These organ dysfunctions are generally known as described inpublications published before the application date of this application,for example, Funada H., “Elucidation and Treatment Strategy for Sepsis”,Iyaku Journal Co., Ltd., p. 38—(2006)), “Surviving Sepsis Campaign:international guidelines for management of severe sepsis and septicshock 2008” (Crit. Care Med., 2008 January; 36(1):296-327), and thelike.

It is supposed that organ dysfunction may be induced by a factor otherthan sepsis, such as in the case of drug-induced organ dysfunction, andaccordingly, it is desirable that patients with organ dysfunctionlimited to the liver or kidney are excluded from the severe septicpatients. It is also known that thrombocytopenia may be developed as aresult of organ dysfunction. Although platelet count in patients to beadministered with the medicament of this embodiment is not particularlylimited so far that the platelet count is less than 300,000/μL, thecount is preferably less than 200,000 μL, more preferably less than150,000/μL.

In this embodiment, the value of INR in a plasma specimen of a sepsispatient is not particularly limited so far that the value is more than1.4, and when INR is more than 1.4, thrombomodulin is more effective forsepsis patients with one or more organ dysfunctions. The maximum INR maybe, for example, 2.0 or lower, preferably 1.9 or lower, more preferably1.8 or lower, still more preferably 1.7 or lower, most preferably 1.6 orlower. The value may also be preferably 1.5 or lower. It may also bepreferred that a patient with an INR value of 1.7 is excluded.

The expression “INR more than 1.4” may also be indicated as “INR>1.4”.

In this embodiment, DIC is a disease or syndrome whereby largequantities of blood coagulation-accelerating substances are generated asa result of tissue damage caused by various diseases, so that thefunction of a coagulation system is excessively accelerated, and smallthrombuses are formed in generalized blood vessel (microthrombusformation) and they clog small vessels, and at the same time,thrombocytes or coagulation factors necessary for the control ofbleeding are consumed, thereby causing clotting abnormality.Specifically, as a result of fibrin formation in vascular vessel,bleeding due to consumption coagulopathy or organ failure due tomicrothrombus formation occurs. DIC is also referred to as disseminatedintravascular coagulation syndrome or diffuse intravascular coagulationsyndrome.

DIC has various types of clinical symptoms depending on the type ofunderlying pathogenic condition. In addition to observation of bleedingor organ symptoms, a preferred method for diagnosing an illness as DICcomprises keeping the score of DIC on the basis of several test valuesas described below and then diagnosing the illness as DIC when the DICscore has reached a certain level. Examples of such test values includethe number of blood platelets, the concentration of fibrin/fibrinogendegradation products (hereinafter abbreviated as FDP, at times)decomposed by plasmin, a D-dimer concentration, a fibrinogenconcentration, and a prothrombin time. Moreover, it is also possible todiagnose a certain condition as preDIC based on a decrease in platelets,an increase in the D-dimer or FDP concentration, and the like withoutkeeping the DIC score (Masao Nakagawa, “Search report regarding use ofcriteria of disseminated intravascular coagulation (DIC),” ResearchStudy Team of Intractable Disease (Blood Coagulation Abnormality), theMinistry of Health and Welfare, Study report 1999, 1999: 65-72; KatsumiDeguchi, “Tentative plan regarding standards for initiation of earlytreatment of DIC,” Research Study Team of Intractable Disease (BloodCoagulation Abnormality), the Ministry of Health and Welfare, Studyreport 1999, 1999: 73-77; and Katsumi Nakagawa & Hajime Tsuji, “Currentdiagnosis of DIC—Reports on results of inquiry survey” Clinical Blood.1999, 40: 362-364).

In this embodiment, a sepsis patient with an INR value more than 1.2,preferably more than 1.3, more preferably more than 1.4, in a plasmaspecimen of the patient can be called a DIC patient in a broad sense,and the medicament for therapeutic treatment and/or improvement ofsepsis according to this embodiment may be used as a medicament fortherapeutic treatment and/or improvement of DIC.

The medicament of this embodiment may also be used for DIC. Sepsis isalso regarded as SIRS induced by critical clinical invasion frominfection, and closely relates to DIC of which causative disease is aninfectious disease. DIC is often developed simultaneously with sepsis,and the medicament of this embodiment may also be used for such a sepsispatient simultaneously developing DIC. In other words, the medicament ofthis embodiment may be used for a patient suffering from or suspected tosuffer from either one of DIC and sepsis, or the both.

In this embodiment, INR can be measured, for example, as follows.Specifically, tissue thromboplastin and Ca²⁺ are added to plasma (testspecimen) obtained by adding sodium citrate, time (PT) required forcoagulation (precipitation of fibrin) is measured, and evaluation isestablished on the basis of relative ratio of the time in terms ofsecond with respect to that of a control specimen (activity ratio). Theactivity ratio can be obtained as “coagulation time (second) of testspecimen/coagulation time (second) of control specimen”, but the ratiomay vary among laboratories in which the test is implemented due todifference in sensitivity of used tissue thromboplastin. The INR valuewas devised in order to eliminate such variation, and by evaluating PTusing the INR value corrected with an international sensitivity index(henceforth also abbreviated as ISI), variation caused by difference oflaboratories can be eliminated to obtain a standard result. ISIrepresents difference from the international standard sample. ISI isdetermined for every tissue thromboplastin reagent, and is attached tothe reagent. Examples of the thromboplastin reagent include Thromborel S(registered trademark, Sysmex Corp.), Thromboplastin C+ (registeredtrademark, Sysmex Corp.), and the like, but not limited to theseexamples. Thromborel S (registered trademark) uses human placentathromboplastin (ISI value is around 1.0), and thromboplastin C+(registered trademark) uses rabbit brain thromboplastin (ISI value isabout 1.8).

ISI is attached to each tissue thromboplastin reagent, and the INR valueis calculated in accordance with Equation 1 mentioned above.

Although the control specimen is not particularly limited so far thatthe specimen is a commercially available pooled normal human plasma,there can be used commercially available pooled citrated (Na citrate)normal human plasma, and the like, available from, for example,Kojin-Bio Co., Ltd. or International Bioscience Inc.

As therapeutic treatments of sepsis, such basic treatments as mentionedbelow are generally performed by referring to known publications(Surviving Sepsis Campaign: International guidelines for management ofsevere sepsis and septic shock: Crit. Care Med., 2008, 36:296-327; Crit.Care Med., 32(3), 1250-56 (2003)), and thrombomodulin and anothermedicament may be used in combination. However, the other medicamentused in combination is not limited to those mentioned below.

When hypotension continues in a septic shock patient even after thecentral venous pressure (CVP) rises to a desired value, dopamine may beadministered in order to raise the average blood pressure to at least 60mmHg. When the dopamine dose exceeds 20 μg/kg/minute, anothervasopressor (usually norepinephrine) may be additionally administered.

For therapeutic treatment against causative bacteria of sepsis, anantibiotic is generally used. For the selection of the antibiotic, thereis required well-grounded estimation based on suspected cause, clinicalsign, knowledge concerning microorganisms and knowledge concerningpattern of sensitivity common to a specific hospital ward forinpatients, results of preliminary culture test, and the like. Intensivenormalization of blood sugar level in sepsis patients improves clinicaloutcome of the patients in critical conditions.

When an antibiotic is used, a specimen such as blood, body fluid orwound part can be investigated, and a drug effective for the causativebacterium can be chosen. For example, in the case of septic shock ofunknown cause, gentamycin or tobramycin and a third generationcephalosporin may be administered in combination. Further, wheninfection of resistant Staphylococcus or Enterococcus bacteria issuspected, vancomycin is additionally administered.

In general, the dose is adjusted to maintain the blood sugar level at 80to 110 mg/dL (4.4 to 6.1 mmol/L) by continuous intravenous injection ofinsulin.

Since corticosteroid therapy is effective for the therapeutic treatmentof sepsis, it may be administered at a supplemental dose.

To a patient of high death risk (APACHE II score ≧25, multiple organfailure due to sepsis, ARDS due to septic shock or sepsis), arecombinant activated Protein C (rhAPC, drotrecogin α) may beadministered when there are no contraindications (hemorrhage and thelike).

Although target patients are limited, packed red blood cell transfusionmay be performed aiming at Hb 7.0 to 9.0 g/dL.

In the case of impaired erythropoiesis in a sepsis patient due to renalfailure, erythropoietin (EPO) may be administered.

In the case of severe sepsis, heparin may be administered at a low doseunfractionated heparin or low molecular weight heparin may beadministered for prevention of DVT.

The medicament of the present invention may contain a carrier. As thecarrier usable in the present invention, a water-soluble carrier ispreferred, and tonicity agent, buffering agent, viscosity enhancer,surfactant, preservative, antiseptic, soothing agent, pH modifier, orthe like acceptable as pharmaceutical additives is usually preferred.For example, the medicament of the present invention can be prepared byadding sucrose, glycerin, pH modifier consisting of an inorganic salt,or the like as additives. Further, if necessary, amino acids, salts,carbohydrates, surfactants, albumin, gelatin or the like may be added asdisclosed in Japanese Patent Unexamined Publication Nos. 64-6219 and6-321805. Method for adding these additives is not particularly limited.However, in the case of preparing a lyophilized product, examplesinclude, for example, a method of mixing a solution containing at leastone therapeutic agent selected from an immunosuppressant and atherapeutic agent for hematological malignancy, and a solutioncontaining thrombomodulin, then adding additives to the mixture, andmixing the resulting mixture, and a method of mixing additives with atleast one therapeutic agent selected from an immunosuppressant and atherapeutic agent for hematological malignancy dissolved in water, waterfor injection, or an appropriate buffer beforehand, adding a solutioncontaining thrombomodulin to the mixture, mixing the resulting mixtureto prepare a solution, and lyophilizing the solution, in manners asthose commonly employed. When the medicament of the present invention isa medicament comprising a combination of the components of themedicament, each component is preferably prepared by adding a carrieraccording to an appropriate preparation method. The medicament of thepresent invention may be provided in the form of an injection, or in theform of a lyophilized preparation to be dissolved upon use.

As for preparation of the medicament of the present invention, anaqueous solution for injection can be prepared by filling a solutioncontaining 0.1 to 10 mg of thrombomodulin, water for injection, andadditives in an ampoule or vial in a volume of, for example, 0.5 to 10mL. Examples of the preparation method also include a method of freezingsuch a solution, and drying the frozen solution under reduced pressureto prepare a lyophilized preparation.

The medicament of the present invention is desirably administered byparenteral administration such as intravenous administration,intramuscular administration, and subcutaneous administration. Themedicament may also be administered by oral administration, intrarectaladministration, intranasal administration, sublingual administration orthe like. When the medicament of the present invention is a medicamentcomprising a combination of multiple active ingredients, each activeingredient of the medicament is preferably administered by anadministration method suitable for the ingredient.

Examples of method for the intravenous administration include a methodof administering a desired dose of the medicament at one time(intravenous bolus administration), and intravenous administration bydrip infusion.

The method of administering a desired dose of the medicament at one time(intravenous bolus administration) is preferred from the viewpoint thatthe method requires only a short time for administration. Especially itis preferred in case of sepsis patients who need the urgent treatment.When the medicament is administered at one time, a period required foradministration by using an injectable syringe may generally varies. Ingeneral, the period of time required for the administration is, forexample, 5 minutes or shorter, preferably 3 minutes or shorter, morepreferably 2 minutes or shorter, still more preferably 1 minute orshorter, particularly preferably 30 seconds or shorter, although itdepends on a volume to be administered. Although the minimumadministration time is not particularly limited, the period ispreferably 1 second or longer, more preferably 5 seconds or longer,still more preferably 10 seconds or longer. The dose is not particularlylimited so long that the dose is within the aforementioned preferreddose. Intravenous administration by drip infusion is also preferred froma viewpoint that blood level of thrombomodulin can be easily keptconstant.

A daily dose of the medicament of the present invention may varydepending on age, body weight of patients, severity of disease,administration route and the like. In general, the maximum dose ispreferably 20 mg/kg or less, more preferably 10 mg/kg or less, stillmore preferably 5 mg/kg or less, particularly preferably 2 mg/kg orless, and most preferably 1 mg/kg or less, and the minimum dose ispreferably 0.001 mg/kg or more, more preferably 0.005 mg/kg or more,still more preferably 0.01 mg/kg or more, particularly preferably 0.02mg/kg or more, and most preferably 0.05 mg/kg or more, in terms of theamount of thrombomodulin.

In the case of intravenous bolus administration, although the dose isnot particularly limited so long as the dose is within theaforementioned preferred dose, the maximum daily dose is preferably 1mg/kg or less, more preferably 0.5 mg/kg or less, still more preferably0.1 mg/kg or less, particularly preferably 0.08 mg/kg or less, and mostpreferably 0.06 mg/kg or less, and the minimum dose is preferably 0.005mg/kg or more, more preferably 0.01 mg/kg or more, still more preferably0.02 mg/kg or more, and particularly preferably 0.04 mg/kg or more.

When the medicament of the present invention is administered to apatient having a body weight exceeding 100 kg, it may be preferablyadministered at a fixed dose of 6 mg, since blood volume is notproportional to the body weight, and blood volume is relatively reducedwith respect to the body weight in such a patient.

In the case of continuous intravenous infusion, although the dose is notparticularly limited so long as the dose is within the aforementionedpreferred dose, the maximum daily dose is preferably 1 mg/kg or less,more preferably 0.5 mg/kg or less, still more preferably 0.1 mg/kg orless, particularly preferably 0.08 mg/kg or less, and most preferably0.06 mg/kg or less, and the minimum dose is preferably 0.005 mg/kg ormore, more preferably 0.01 mg/kg or more, still more preferably 0.02mg/kg or more, and particularly preferably 0.04 mg/kg or more.

When the medicament of the present invention is administered to apatient having a body weight exceeding 100 kg, it may be preferablyadministered at a fixed dose of 6 mg, since blood volume is notproportional to the body weight, and blood volume is relatively reducedwith respect to the body weight in such a patient.

The medicament is administered once or several times a day as required.As for administration interval, the medicament may be administered oncein 2 to 14 days, preferably once in 2 to 7 days, more preferably once in3 to 5 days.

[Explanation of Sequence Listing]

SEQ ID NO: 1: Amino acid sequence encoded by the gene used in productionof TME456SEQ ID NO: 2: Nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 1SEQ ID NO: 3: Amino acid sequence encoded by the gene used in productionof TME456MSEQ ID NO: 4: Nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 3SEQ ID NO: 5: Amino acid sequence encoded by the gene used in productionof TMD12SEQ ID NO: 6: Nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 5SEQ ID NO: 7: Amino acid sequence encoded by the gene used in productionof TMD12MSEQ ID NO: 8: Nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 7SEQ ID NO: 9: Amino acid sequence encoded by the gene used in productionof TMD123SEQ ID NO: 10: Nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 9SEQ ID NO: 11: Amino acid sequence encoded by the gene used inproduction of TMD123MSEQ ID NO: 12: Nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 11SEQ ID NO: 13: Synthetic DNA for mutation used for carrying outsite-directed mutagenesis

EXAMPLES

The present invention will be explained in detail with reference toexamples and test examples. However, the present invention is notlimited by these examples.

The thrombomodulin of the present invention used in the test exampleswas prepared according to the aforementioned method of Yamamoto et al.(the method described in Japanese Patent Unexamined Publication No.64-6219). Preparation examples thereof are described below. Safety ofthe thrombomodulins obtained in these preparation examples was confirmedby single and repetitive intravenous administration tests using rats andmonkeys, mouse reproduction test, local irritation test, pharmacologicalsafety test, virus inactivation test, and the like.

Preparation Example 1 Obtaining Thrombomodulin

A highly purified product was obtained by the aforementioned method.Specifically, Chinese hamster ovary (CHO) cells were transfected with aDNA encoding the amino acid sequence of SEQ ID NO: 9 (which specificallyconsisted of the nucleotide sequence of SEQ ID NO: 10). From the cultureof the above transformant cells, a highly purified product was obtainedby collecting an active fraction with a 20 mmol/L phosphate buffer (pH7.3) containing 50 mmol/L NaCl according to the aforementionedconventional purification method. The product was further concentratedby using an ultrafiltration membrane to obtain a thrombomodulin solutionhaving a concentration of 11.0 mg/mL (henceforth also abbreviated asTMD123 in the specification).

<Preparation of Polysorbate Solution>

Polysorbate 80 was weighed (0.39 g) in a glass beaker, added with waterfor injection (30 mL), and dissolved.

<Preparation and Filling of Drug Solution>

The TMD123 solution obtained above (2239 mL, corresponding to 24.63 g ofsoluble thrombomodulin protein, added in a 5% excess amount) was putinto a 5-L stainless steel vessel. The polysolvate solution obtainedabove was further added, and sodium chloride (27.9 g) was added. Waterfor injection (600 mL) was added, and the mixture was stirred. Themixture was adjusted to pH 6.0 by adding a 1 mol/L hydrochloric acidsolution. Water for injection was further added to the mixture up to atotal amount of 3940 g, and the mixture was uniformly mixed and stirred.This drug solution was subjected to filtration sterilization using afilter having a pore diameter of 0.22 μm (MCGL10S, manufactured byMillipore). The filtrate was filled in ampoules in an amount of 1.1 geach to obtain a TMD123 preparation.

Preparation Example 2

Chinese hamster ovary (CHO) cells are transfected with a DNA encodingthe amino acid sequence of SEQ ID NO: 11 (which specifically consists ofthe nucleotide sequence of SEQ ID NO: 12), a solution of thrombomodulinpurified from a culture of the above transformant cells (henceforth alsoabbreviated as TMD123M in the specification) by the aforementionedconventional purification method is obtained, and a TMD123M preparationis obtained in the same manner as that described above.

Preparation Example 3

Chinese hamster ovary (CHO) cells are transfected with a DNA encodingthe amino acid sequence of SEQ ID NO: 1 (which specifically consists ofthe nucleotide sequence of SEQ ID NO: 2), thrombomodulin purified from aculture of the above transformant cells (henceforth also abbreviated asTME456 in the specification) by the aforementioned conventionalpurification method is obtained, and a TME456 preparation is obtained inthe same manner as that described above.

Preparation Example 4

Chinese hamster ovary (CHO) cells are transfected with a DNA encodingthe amino acid sequence of SEQ ID NO: 3 (which specifically consists ofthe nucleotide sequence of SEQ ID NO: 4), thrombomodulin purified from aculture of the above transformant cells (henceforth also abbreviated asTME456M in the specification) by the aforementioned conventionalpurification method is obtained, and a TME456M preparation is obtainedin the same manner as that described above.

Preparation Example 5

Chinese hamster ovary (CHO) cells are transfected with a DNA encodingthe amino acid sequence of SEQ ID NO: 5 (which specifically consists ofthe nucleotide sequence of SEQ ID NO: 6), thrombomodulin purified from aculture of the above transformant cells (henceforth also abbreviated asTMD12 in the specification) by the aforementioned conventionalpurification method is obtained, and a TMD12 preparation is obtained inthe same manner as that described above.

Preparation Example 6

Chinese hamster ovary (CHO) cells are transfected with a DNA encodingthe amino acid sequence of SEQ ID NO: 7 (which specifically consists ofthe nucleotide sequence of SEQ ID NO: 8), thrombomodulin purified from aculture of the above transformant cells (henceforth also abbreviated asTMD12M in the specification) by the aforementioned conventionalpurification method is obtained, and a TMD12M preparation is obtained inthe same manner as that described above.

Preparation Example 7 Preparation of Placebo Formulation <Preparation ofPolysorbate Solution>

Polysorbate 80 was weighed (0.4 g) in a glass beaker, added with waterfor injection (30 mL), and dissolved.

<Preparation and Filling of Drug Solution>

Water for injection (2000 mL) was put into a 5-L stainless steel vessel.The polysolvate solution obtained above was further added. Water forinjection was further added to the mixture up to a total amount of 4000g, and the mixture was uniformly mixed and stirred. This drug solutionwas subjected to filtration sterilization using a filter having a porediameter of 0.22 μm (MCGL10S, manufactured by Millipore). The filtratewas filled in ampoules in an amount of 1.1 g each to obtain a placebopreparation.

Example 1 Method for Experiment

By using TMD-123 prepared according to Preparation Example 1 asthrombomodulin, a randomized double-blind placebo-controlled study wasconducted for patients with sepsis and DIC. Targeted patient number is750 in total, and among them, 741 patients were administered withinvestigational drug (TMD-123 for 370 patients and placebo for 371patients). TMD-123 was administered once a day at 0.06 mg/kg forsuccessive 6 days via intravenous bolus administration. As the placebo,the preparation manufactured according to Preparation Example 7 wasused.

For patients over the body weight of 100 kg, a fix dose of 6 mg wasevenly administered once a day for successive 6 days via intravenousbolus administration in order to suppress side effects due to overdose.

A plasma INR value of a patient before the administration of the testdrug was measured by the above method described as Equation 1.

Severe septic patients with organ dysfunction limited to the liver orkidney were excluded for the analysis. In patients with organdysfunction limited to the liver or kidney, the organ dysfunction maypossibly be occurred on the basis of causes not limited to sepsis suchas drug-induced organ dysfunction.

Outcome after 28 days from the start of the administration was observedand a mortality rate (Mortality) of each patent group was calculated.

Further, a difference in mortality rate between TMD-123 group andplacebo group was calculated as “Difference”.

<Study Results>

In the group of patient without organ dysfunction, patients with the INRvalue of INR>1.5 in plasma before the administration of the test druggave maximum difference (Difference: 6.1%) between the mortality rate ofthe group administered with TMD-123 and that of the group administeredwith placebo, and the group of patient with INR>1.6 gave the secondhighest difference in mortality rate (4.5%). In the group of patientwith INR>1.4, no significantly high difference in mortality rate wasabsolutely or relatively observed, i.e., difference in mortality ratewas about 1.7% (Table 1).

From Table 1, it is recognized that the maximum result can be foundbetween 1.5 and 1.6 of the lower limits of INR, and when the lowerlimits of INR are outside the range of from 1.5 to 1.6, the differencein mortality rate was significantly decreased.

Whilst, in the group of severe septic patients having one or more organdysfunctions selected from circulatory organ dysfunction, respiratoryorgan dysfunction, kidney dysfunction, and liver dysfunction, the groupof patients with INR>1.4, not the group with INR>1.5, gave the maximumdifference in mortality rate (9.7%), which is absolutely and relativelyhigh difference in mortality rate as compared to the other INR lowerlimits (Table 2). The group of severe septic patients having one or moreorgan dysfunctions selected from circulatory organ dysfunction,respiratory organ dysfunction, kidney dysfunction, and liver dysfunctiongave more significant difference in mortality rate between TMD-123 groupand placebo group on the whole as compared to the group of patientswithout organ dysfunction (the former: 5.4%, the latter −1.1%).

Further, in the group of severe septic patients having one or more organdysfunctions selected from circulatory organ dysfunction, respiratoryorgan dysfunction, kidney dysfunction, and liver dysfunction, thepatient with 1.4<INR≦1.6 gave absolutely remarkable difference inmortality rate as being 16.0% (Table 3). As understood from Table 3, anyof the other INR upper limits gave values of the difference in mortalityrate in a range of 10 to 12%, which indicates relatively recognizableremarkably high difference in mortality rate of the aforementionedgroup.

On the other hand, no tendency of relatively outstanding peak increasein a certain group was observed in the group of patients without organdysfunction. The difference in mortality rate obtained was found atutmost to be 7.1% (in patient group with 1.4<INR≦1.7) (Table 4).

TABLE 1 INR value of plasma before TMD-123 Placebo administration NumberMortality Number Mortality Difference INR > 1.2 106 10/106 = 9.4% 939/93 = 9.7% 0.2% INR > 1.3 81   6/81 = 7.4% 75 6/75 = 8.0% 0.6% INR >1.4 71   5/71 = 7.0% 69 6/69 = 8.7% 1.7% INR > 1.5 57   3/57 = 5.3% 53 6/53 = 11.3% 6.1% INR > 1.6 42   3/42 = 7.1% 43  5/43 = 11.6% 4.5%INR > 1.7 29    3/29 = 10.3% 27 1/27 = 3.7% −6.6%   INR > 1.8 22    3/22= 13.6% 20 1/20 = 5.0% −8.6%   INR > 1.9 14    2/14 = 14.3% 12 14/32 =8.3%  −6.0%   INR > 2.0 9     1/9 = 11.1%  7   1/7 = 14.3% 3.2%

TABLE 2 INR value of plasma before TMD-123 Placebo administration NumberMortality Number Mortality Difference INR > 1.2 184 44/184 = 23.9% 18655/186 = 29.6% 5.7% INR > 1.3 160 40/160 = 25.0% 162 52/162 = 32.1% 7.1%INR > 1.4 137 33/137 = 24.1% 136 46/136 = 33.8% 9.7% INR > 1.5 10126/101 = 25.7% 100 34/100 = 34.0% 8.3% INR > 1.6 81  23/81 = 28.4% 77 26/77 = 33.8% 5.4% INR > 1.7 61  19/61 = 31.1% 65  25/65 = 38.5% 7.3%INR > 1.8 45  15/45 = 33.3% 48  19/48 = 39.6% 6.3% INR > 1.9 36  14/36 =38.9% 32  14/32 = 43.8% 4.9% INR > 2.0 27  13/27 = 48.1% 25  14/25 =56.0% 7.9%

TABLE 3 INR value of plasma before TMD-123 Placebo administration NumberMortality Number Mortality Difference 1.6 >= INR > 1.4 56  10/56 = 17.9%59  20/59 = 33.9% 16.0% 1.7 >= INR > 1.4 76  14/76 = 18.4% 71  21/71 =29.6% 11.2% 1.8 >= INR > 1.4 92  18/92 = 19.6% 88  27/88 = 30.7% 11.1%1.9 >= INR > 1.4 101 19/101 = 18.8% 104 32/104 = 30.8% 12.0% 2.0 >=INR > 1.4 110 20/110 = 18.2% 111 32/111 = 28.8% 10.6%

TABLE 4 INR value of plasma before TMD-123 Placebo administration NumberMortality Number Mortality Difference 1.6 >= INR > 1.4 29 2/29 = 6.9% 261/26 = 3.8% −3.1%   1.7 >= INR > 1.4 42 2/42 = 4.8% 42  5/42 = 11.9%7.1% 1.8 >= INR > 1.4 49 2/49 = 4.1% 49  5/49 = 10.2% 6.1% 1.9 >= INR >1.4 57 3/57 = 5.3% 57 5/57 = 8.8% 3.5% 2.0 >= INR > 1.4 62 4/62 = 6.5%62 5/62 = 8.1% 1.6%

INDUSTRIAL APPLICABILITY

The medicament of the present invention containing thrombomodulin isuseful as a medicament enabling effective therapeutic treatment and/orimprovement of sepsis in a severe septic patient, wherein a INR value ofa plasma specimen obtained from said patient is more than 1.4.

1-9. (canceled)
 10. A method for therapeutic treatment of sepsis, whichcomprises the step of: intravenously administering thrombomodulin to apatient with severe sepsis accompanied with one or more organdysfunctions selected from the group consisting of respiratory organdysfunction and circulatory organ dysfunction, wherein a value ofInternational Normalized Ratio (INR) of a plasma specimen obtained fromsaid patient is more than 1.4, wherein said thrombomodulin isadministered at a dose of 0.04-0.08 mg/kg/day for 6 days.